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The Interplay between the Glucocorticoid Receptor and Nuclear Factor-B or Activator Protein-1: Molecular Mechanisms for Gene Repression

Department of Molecular Biology, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium

Correspondence: Address all correspondence and requests for reprints to: Prof. Guy Haegeman, Department of Molecular Biology, University of Gent, K. L. Ledeganckstraat 35, Gent, Belgium 9000. E-mail:

	Abstract

Top Abstract I. Introduction III. General Conclusion References

 
The inflammatory response is a highly regulated physiological process that is critically important for homeostasis. A precise physiological control of inflammation allows a timely reaction to invading pathogens or to other insults without causing overreaction liable to damage the host. The cellular signaling pathways identified as important regulators of inflammation are the signal transduction cascades mediated by the nuclear factor-B and the activator protein-1, which can both be modulated by glucocorticoids. Their use in the clinic includes treatment of rheumatoid arthritis, asthma, allograft rejection, and allergic skin diseases. Although glucocorticoids have been widely used since the late 1940s, the molecular mechanisms responsible for their antiinflammatory activity are still under investigation. The various molecular pathways proposed so far are discussed in more detail.

I. Introduction

A. NF-B

B. AP-1

C. Glucocorticoid (GC) hormones

II. Molecular Mechanisms

A. GC receptor (GR) activity and direct DNA binding

B. Protein-protein cross-talk

C. Up-regulation of IB-

D. Cofactor competition model

E. New perspectives

III. General Conclusion

	I. Introduction

Top Abstract I. Introduction III. General Conclusion References

 
THE INFLAMMATION PROCESS was first described by Cornelius Celsus (30 BC–38 AD) who mentioned that "rubor et tumor cum calore et dolore" (redness and swelling, accompanied with heat and pain) are the cardinal symptoms of inflammation. The inflammatory response can be interpreted as notification of a threatening agent or organism and subsequent activation of the defense system developed to eliminate these threats. Immunity and inflammation are physiological processes of profound importance to the organism; without these processes, a host would quickly succumb to invading pathogens or damaging stimuli, whereas excessive or inappropriate activation of these responses causes tissue and cell damage and even death. Therefore, maintaining immune homeostasis is critical for the survival of an organism. Both pro- and antiinflammatory mechanisms must be present and functional for a cell (organism) to survive in the face of environmental stimuli that elicit an immune response. These pathways provide homeostasis by pulling the cell in opposite directions (1, 2, 3, 4). Over the last 10 yr, the transcription factors nuclear factor (NF)-B and activator protein (AP)-1 have been shown to be crucial for the induction of genes involved in inflammation, as well as in a wide range of diseases originating from chronic activation of the immune system, including asthma, atherosclerosis, inflammatory bowel disease, and autoimmune diseases such as multiple sclerosis and rheumatoid arthritis (5, 6, 7, 8). A plethora of immunoregulatory genes coding for cytokines, cytokine receptors, chemotactic proteins, or adhesion molecules, such as TNF-, IL-1ß, IL-2, IL-6, IL-8, macrophage chemotactic protein (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), interferon (IFN)-ß, granulocyte-macrophage colony stimulating factor (GM-CSF), intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and E-selectin, contain NF-B and/or AP-1 sites in their promoters or regulatory regions. Therefore, both transcription factors represent an obvious target for immunosuppressive therapies (9, 10, 11, 12, 13, 14, 15). Glucocorticoids (GCs) and catecholamines, the major stress hormones, counteract the production of (pro)inflammatory cytokines, such as IL-12, IL-6, and TNF-, whereas they stimulate the production of antiinflammatory cytokines such as IL-10, IL-4, and TGF-ß (16, 17, 18, 19). Systemically, by activation of the stress system, an excessive immune response stimulates an important negative feedback mechanism, which protects the organism from an overshoot of proinflammatory cytokines and other tissue-damaging products (3, 20, 21, 22, 23, 24).

A. NF-B
Transcriptional regulators of the NF-B/inhibitory protein (I)B family promote expression of more than 100 target genes, the majority of which participate in the host immune response (4, 25, 26, 27, 28) (for a recent update, visit http://people. bu.edu/gilmore/nf-kb/). Gene knockout and other studies established roles for NF-B in the ontogeny of the immune system and demonstrated that NF-B participates at multiple steps during oncogenesis and regulation of programmed cell death (5, 8, 29, 30, 31). The involvement of the ubiquitous transcription factor NF-B in the pathogenesis of the inflammatory response has been well documented by experiments, both in vitro and in vivo (5, 6, 7, 10, 32). NF-B is a heterodimer, typically consisting of p50 and p65 monomeric proteins. A targeted disruption of the genes encoding p50 or p65 leads to extreme immunodeficiencies, and even to lethality in the case of p65 knockout mice (28, 33, 34). The mammalian NF-B/Rel family includes five members: p65 or RelA, RelB, c-Rel, NF-B1 (p50/p105), and NF-B2 (p52/p100). All members are characterized by a conserved stretch of 300 amino acids, designated as the Rel homology domain (RHD). This domain is important for DNA binding and mutual interactions between the different Rel family members. It also serves as an interaction surface for the IB. NF-B is latently present in the cytoplasm, under tight control of the associated protein IB-. The IB protein family comprises the following members: IB-, IB-ß, IB-/p105, IB-/p100, IB-, and B cell lymphoma (Bcl)-3. They are characterized by several 30- to 33-amino acid motifs called ankyrin repeats. Potent inducers of NF-B include the proinflammatory cytokines IL-1 and TNF, byproducts of microbial, fungal, and viral infections such as lipopolysaccharides (LPS), sphingomyelinase, double strand (ds)RNA, Tax protein from human T cell leukemia/lymphoma virus (HTLV), and proapoptotic and necrotic stimuli, such as oxygen-free radicals, UV irradiation, and irradiation. The first step in the activation process of NF-B is an IB kinase complex (IKK)-dependent phosphorylation of IB- at serines 32 and 36. Subsequently, ubiquitinylation at lysines 21 and 22 takes place by a specific ubiquitin ligase belonging to the SCF (Skp-1/Cul/F box) family and tags IB- for degradation by the 26S proteasome complex. The actual recognition of N-terminally phosphorylated IBs is carried out by a WD repeat- and F box-containing protein called ß-TrCP (35). This finally leads to release of the NF-B protein, which migrates to the nucleus to exert its effects on gene regulation (25, 27, 35, 36, 37, 38, 39, 40, 41, 42). Many groups focused on the identification of the serine-specific IB kinase complex IKK, which comprises multiple subunits (43, 44) and acts as an integrator of multiple NF-B-activating stimuli (41, 45, 46).

The differential activity of the two IKK kinases on different IB family members probably also results in a differentially regulated downstream NF-B response and activity (47). Further examination of these proteins confirmed the involvement of IKK-ß in proinflammatory cytokine-induced activation of NF-B, whereas IKK- was found to be crucial for B cell maturation, formation of secondary lymphoid organs, increased expression of certain NF-B target genes, processing of the NF-B2 (p100) precursor, and NF-B activation in the limb and skin during embryogenesis (48, 49, 50). Results from IKK- and IKK-ß double-deficient mice confirmed the importance of IKKs for NF-B activation in vivo and further demonstrated a neuroprotective role for these kinases during development (51). Antagonistic effects of IKK- and IKK-ß have recently also been described in Wnt signaling depending on ß-catenin phosphorylation and localization, thus integrating signaling events between the NF-B and Wnt pathways (52). A third component, IKK- (also known as NEMO/IKKAP/FIP-3), was designated as a scaffold platform for the assembly of the IKK complex (53, 54, 55, 56). Several studies indicate that the IKK complex consists of two IKK-/IKK-ß heterodimers held together by one IKK- monomer. Many proteins have been reported to activate the IKK complex, but so far there is no full understanding of their specificity and redundancy; they include protein kinase (PK)C isozymes, MAPK kinase kinase (MAPKKK) family members, NIK, AKT/TPB, MEKK-1, MEKK-2, MEKK-3, COT/TPL-2, TAK-1 and NAK (46, 57, 58). Many of the previous reports regarding the ability of kinases to activate IKK and induce NF-B DNA binding activity may be the result of overexpression studies and have not necessarily been confirmed by knockout studies (59).

Alternative IKK complexes causing NF-B activation were also identified (42, 60, 61). Besides the classical IB metabolism, variations have also been described at the level of phosphorylation (Ser32, Ser36, Thr273, Tyr42) and degradation (nonproteosomal, lysosomal, or caspase-dependent) (62, 63, 64, 65, 66, 67, 68, 69, 70, 71). Both the release and activity of NF-B are subject to different control mechanisms. IB- expression itself is controlled by NF-B, establishing an autoregulatory feedback loop and shutting down activation of NF-B (72). Furthermore, NF-B activation can be negatively regulated by a SUMO-1 (small ubiquitin-like modifier-1 or sentrine) modification of unphosphorylated IB-. This leads to a degradation-resistant IB molecule (73) which may relocalize to particular subcellular compartments (74). Another level of regulation of NF-B is imposed by the catalytic subunit of PKA, which has been demonstrated to form a cytosolic complex together with NF-B and IB (75). p65 phosphorylation by PKA at Ser276 affects its transcriptional activity and was reported to mediate a functional interaction of NF-B with the cofactor cAMP response element-binding protein (CREB)-binding protein (CBP) (76, 77). Phosphorylation at various other amino acid residues in p65 was also found to contribute to the transcriptional activity of NF-B (28, 42, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86).

B. AP-1
The transcription factor AP-1 is encoded by protooncogenes and regulates various aspects of cell proliferation and differentiation (12, 14, 87). AP-1 can be composed of either homodimers or heterodimers between members of the Jun (c-Jun, v-Jun, Jun-B, and Jun-D), Fos (c-Fos, Fos-B, Fra-1, and Fra-2), activating transcription factor (ATF-2, ATF-3/LRF-1, B-ATF, JDP-1, JDP-2) or Maf (v-Maf, c-Maf, Maf-A/B/F/G/K, Nrl) families; they all belong to the class of the basic zipper (bZIP) family of sequence-specific dimeric DNA-binding proteins. The protein products of the fos and jun gene families, i.e., the so-called immediate-early genes that are directly activated and require no new transcription or translation for their induction, are transcription factors that activate and repress other genes, thereby producing secondary transcriptional reprogramming appropriate to the stimulus used (88, 89, 90). The regulation of AP-1 activity is complex and first occurs by changes in jun and fos gene transcription and mRNA turnover; secondly, by effects on Jun and Fos protein turnover; thirdly, by posttranslational modifications of Jun and Fos proteins that modulate their transactivation potential; and fourthly, by interactions with other transcription factors that can either synergize or interfere with AP-1 activity (12, 88, 89, 90, 91, 92). AP-1 was originally identified to interact with the control regions of genes containing promoter elements responsive to tetradecanoylphorbol acetate. Today, various stimuli, such as physiological agents (growth factors, mitogens, polypeptide hormones, cell-matrix interactions, and inflammatory cytokines), bacterial and viral infections, pharmacological compounds (anisomycin, phorbol esters, and okadaic acid), cellular stress (ultraviolet or ionizing radiation), as well as hyperosmotic and heavy-metal stress, have been shown to induce AP-1 activity. These stimuli activate MAPK cascades [mostly p38, Jun amino-terminal kinase (JNK), and ERKs] that enhance AP-1 activity by phosphorylating distinct substrates. The transcriptional activity of c-Jun is enhanced by amino-terminal phosphorylation at Ser63 and Ser73 by JNK (93). This inducible phosphorylation step is required to recruit the transcriptional coactivator CBP, which leads to transcriptional enhancement (94, 95). In addition to positive regulatory effects, the AP-1 complex has been shown to confer negative regulation, for instance of GC receptor (GR) (96). The growth-promoting activity of c-Jun is mediated by repression of tumor suppressors, as well as up-regulation of positive cell cycle regulators. c-Jun is a mostly positive regulator of cell proliferation, whereas Jun-B has the adverse effect. However, the ability of c-Jun and Jun-B to elicit opposite transcriptional responses in the presence of apparently similar AP-1 recognition sites, found in the control regions of different genes, remains enigmatic (14). Knockout studies indicated a biological role for c-Fos in survival during bone development and homeostasis, gametogenesis, and neuronal functions, besides its role in cell proliferation and differentiation. For c-Jun, a role has also been demonstrated in development, hepatogenesis, and liver erythropoiesis (12).

C. Glucocorticoid (GC) hormones
1. Molecular aspects and physiology.
GCs exert their effects by binding to the GR, a transcription factor capable of regulating several genes in a positive or negative way (for a comprehensive list, see Ref.1). GR belongs to the family of steroid hormone receptors, comprising structurally similar modular proteins, such as GR, progesterone (PR), mineralocorticoid (MR), androgen (AR), and estrogen (ER) receptor forms, which further belong to the nuclear receptor (NR) superfamily (97). Other classes of NRs include thyroid (TR), retinoid and orphan receptors [retinoic acid receptor (RAR)/retinoid X receptor (RXR)]. In general, the receptor members share a variable amino-terminal transactivation domain (98), a central and well-conserved DNA-binding domain (DBD), and a moderately conserved carboxy-terminal domain responsible for ligand binding. The latter domain also contains activating functions (1, 99, 100, 101, 102).

In vivo, GC hormones are synthesized stepwise from cholesterol by a series of cytochrome P450-catalyzed reactions within the adrenal cortex (zona fasciculata). The synthesis and secretion of cortisol, the major GC hormone in man, is tightly controlled by the balance of adrenocorticotropin (secreted from the anterior pituitary gland) and CRH (secreted from the hypothalamus during stress) in a pulsatile and circadian way (103, 104). The most widely accepted mechanism for GC entry into the cell is by free diffusion of the lipophilic molecules across the lipid bilayer of the cell into the cytoplasm. In its unliganded resting state, in the absence of GC hormone, GR is present in the cytoplasm in an inactive complex (i.e., DNA binding-incompetent) with chaperones and cochaperone molecules (105, 106). The most important chaperones in NR action are heat shock protein (hsp)90 and hsp70. Their action is further positively or negatively regulated by cochaperones such as immunophilins (FK506-binding proteins FKBP1/2), dynein, p23, hsp40/hdj1, hip, carboxy terminus of hsp70-interacting protein (CHIP) and BAG-1 (Bcl-2 binding athanogene-1) (105, 107, 108, 109). Receptor activation and hyperphosphorylation occurs upon ligand binding, which initiates substitution of one immunophilin (FKBP-51) for another (FKBP-52), and concomitant recruitment of the transport protein dynein, but leaving hsp90 unchanged. Immunofluorescence and fractionation revealed hormone-induced translocation of the hormone-generated GR-hsp90-FKBP-52-dynein complex from cytoplasm to nucleus, a step that precedes dissociation of the complex within the nucleus and conversion of GR to the DNA-binding form (109, 110). From recent studies, it has become apparent that the role of the (co)chaperones is not only restricted to the cytoplasm. Apart from inhibiting hormone binding to GR, they can also regulate the regulatory functions of the receptors in the nucleus (108) by dynamic (dis)assembly of various transcription complexes (111, 112, 113). Activated GR binds to specific DNA sequences as a homodimer. Genes positively regulated by GR are characterized by GC-response elements (GRE) in the promoter (Fig. 1A and Table 1), whereas negatively regulated genes contain either a negative GRE (nGRE) (Fig. 1B) or are inhibited by direct or indirect interference of GR with the transcriptional activity of other DNA-bound transcription factors [such as NF-B, AP-1, CREB, CCAAT enhancer binding protein (C/EBP), signal transduction activator of transcription (STAT), p53, Smad, etc.] (Fig. 1C–N).

View larger version (39K): [in this window] [in a new window]   FIG. 1. Cartoons of the proposed models as described throughout the text are drawn in Fig. 1 and represented in Table 1, explaining interactions of GR with DNA/transcription factors and corresponding effects on gene regulation (represented by + or - sign). BTM, Basal transcription machinery; nucl., nucleosome; P-TEFb, transcription elongation factor; pol, polymerase; TA, transactivation domain; TF, transcription factor.

3. Tissue specificity of GC effects.
Because GR is expressed in the vast majority of tissues, it is reasonable to assume that GCs affect nearly all cells in the body (130). The regulation and action of GC-mediated effects further depend on other tissue-specific factors, on the bioavailability of the hormone, and on tissue-specific hormone-modifying enzymes. At one level, the biological sensitivity of GCs is achieved by binding to circulating proteins present in plasma and blood, such as corticosteroid-binding globulin (CBG) (131). During a stressful situation (e.g., septic disorder), CBG levels drop due to an IL-6-dependent hepatic posttranscriptional blockade. This results in enhanced exposure of cells and tissues to free GC hormone to suppress the inflammatory response, which would otherwise lead to death. CBG homeostasis is normally restored after 1 or 2 d (132, 133). In kidney, liver, brain, and pancreas cells, 11ß-hydroxysteroid dehydrogenases can convert cortisol to a biologically inactive form or reactivate it from hormone precursors in a cell-specific manner (134, 135, 136). At another level, GC sensitivity is determined by expression levels of the transporter protein LEM1 or multidrug resistance protein MDR1 (137, 138). The expression levels of GR are also cell- and tissue-specific. GR levels are themselves negatively regulated by GCs, contributing to the fact that long-term treatment with GCs results in a decrease of the physiological response (139, 140). Other levels of regulation that determine GC sensitivity include variations in the receptor protein (mutations, polymorphisms, isoforms) (141, 142, 143, 144, 145, 146, 147), alternative receptor dimerization (GR heterodimerization has been described with MR, PR, and AR) (144, 148, 149, 150, 151), presence of GC modulatory element binding proteins (152, 153, 154, 155), receptor cochaperones (111, 112, 156, 157), DNA-bending (158), altered expression levels of hsp proteins (159, 160), effects of signaling cascades (141, 161, 162, 163), and posttranslational modifications (phosphorylation, nitrosylation, ubiquitinylation, sumoylation, and acetylation) (141, 159, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173). Finally, it is now clear that differences between endogenous GCs (produced by the adrenal glands) and synthetic GCs, in terms of their regulatory mechanisms, are crucial for their biological actions. For example, synthetic GCs differ from endogenous GCs in binding to CBG, tissue-specific metabolism, affinity for various GRs, and interaction with transcription factors (174).

4. GCs in the clinic.
GCs belong to the most commonly and effectively used drugs in the clinic to relieve inflammation and various immune disorders (1, 104, 175, 176, 177, 178). Inflammatory diseases, for which administration of GCs are a standard treatment, include rheumatoid arthritis, inflammatory bowel diseases, systemic lupus erythematosus, sarcoidosis, and nephrotic syndrome. Local treatments with GCs are applied against dermatitis, ophthalmological disorders, asthma, and conjunctivitis (179, 180, 181, 182, 183). Furthermore, GCs are used to suppress the immune system post transplantation. GCs are also used to treat brain edema, shock conditions, and certain cancers (e.g., hematological malignancies), as well as conditions involving adrenal cortex insufficiency (e.g., Addison’s disease). There is a huge drawback, however, to the beneficial use of GCs, because treatments with high doses for longer periods cannot only cause resistance to the steroid-based therapy (184, 185), but can also be accompanied by a range of detrimental side-effects (178, 186, 187, 188). These include diabetes, impaired wound healing, skin atrophy, muscle atrophy, increased susceptibility to infections, activation of latent infections, hypothalamus-pituitary-adrenal axis insufficiency, cataracts, peptic ulcers, hypertension (due to activation of the MR), metabolic disorders (resulting from hyperglycemia and a decreased carbohydrate tolerance), retention of water and sodium and excretion of potassium (disturbing the water household balance of the body), and loss of mineral from bone (leading to osteoporosis) (104, 176, 178, 189, 190, 191, 192, 193, 194, 195). To date, physicians attempt to minimize these side-effects with local therapies, intervals, supplementation with calcium, vitamin D3, and estrogens, and using specific GCs with a minimum of mineralocorticoid agonistic effects (178).

5. GCs and inflammation.
GCs have been described to inhibit leukocyte migration to the sites of inflammation and to interfere with the functions of endothelial cells, leukocytes, and fibroblasts. They suppress the production and effects of humoral factors involved in the inflammatory response (104, 196). From a mechanistic point of view, it is generally assumed that the beneficial, antiinflammatory potential of the GR resides in a negative modulation of proinflammatory cytokines and that its side-effects are mainly the consequence of its transactivating capacities (197). Nevertheless, other compounds have not matched the clinical use of GCs as a potent immune suppressive and antiinflammatory agent.

To explain the repressive action of GCs on immune target genes, the role of GCs in inhibiting the activity of the transcription factors NF-B, AP-1, or CREB has been widely investigated. Table 2 lists a number of proinflammatory genes and the main transcription factors contributing to their up-regulation. It would be an improvement for many steroid-treated patients if one could redesign GR function and reduce its side-effects while retaining the antiinflammatory characteristics (198). To that end, many investigators are currently trying to elucidate how GCs exert their mechanism of action (177, 199). The final goal is to reach a more effective and targeted immunosuppressive therapy. In this respect, the development and characterization of so-called dissociating GCs, which separate transrepression from transactivation, have been the holy grail of steroid pharmacology for years, although they did not live up to their expectations in vivo so far (198, 200, 201, 202, 203, 204, 205, 206, 207, 208).

II. Molecular Mechanisms
A. GC receptor (GR) activity and direct DNA binding
Activated GR binds to specific DNA sequences as a homodimer. The dimerization domain (DBD) consists of two zinc ions coordinated with eight cysteine residues to form two zinc fingers. Each zinc finger is followed by an amphipathic -helix. GR DBDs bind cooperatively to specifically spaced target half-sites in the DNA (the consensus sequence is 5'-GGTACAnnnTGTTCT-3'); the N-terminal zinc finger is involved in specific DNA interaction, whereas the C-terminal zinc finger mainly provides DNA-dependent dimerization (209, 210). One function of the DBD is to discriminate between different response elements and determine which target genes are activated. This function is achieved by a few crucial amino acids localized in the C-terminal part of the N-terminal zinc finger, the so-called P-box (211).

Direct transcriptional repression by GCs can be achieved by the interaction of GR with a site on the DNA, designated nGRE, of which the actual sequence is poorly defined. This mechanism of action was proposed to account for repression of the proopiomelanocortin (POMC) gene (precursor of ACTH), type 1 vasoactive intestinal polypeptide (VIPR1), keratin, prolactin (PRL) and proliferin genes, as well as the vitamin D-induced osteocalcin gene (212) (Fig. 1, B and C). Detailed footprinting revealed that the function of nGREs is to instruct GR to bind as a monomer (213). In addition, for some of these genes the mechanism was also found to involve GR-dependent displacement of another factor (for example TATA-binding protein TBP) or DNA-independent tethering by GR of another transcription factor (214, 215) (Fig. 1, D and E). GR tethering of the transcription factors CREB, AP-1, or the orphan NR Nurr-77 has been studied in detail in the human glycoprotein hormone -subunit (216, 217), the collagenase gene (96, 218), and the POMC gene (212, 219), respectively. A variation on this theme is observed for the proliferin gene, in which a composite GRE/AP-1 site, termed pflG, was defined; the GR can regulate activated AP-1 and enhance transcription of proliferin if AP-1 consists of c-Jun homodimers, but represses when AP-1 consists of c-Jun/c-Fos heterodimers (220, 221) (Fig. 1F). A similar regulation was reported for -fetoprotein (222). Finally, a nGRE/Pit1/XTF composite element was detected in the PRL3 gene (223).

B. Protein-protein cross-talk
Because no nGRE could be detected in the majority of inflammatory genes, transcriptional interference was discovered to mostly result from cross-talk between the GR and other transcription factors, such as NF-B or AP-1 (Table 2) (224, 225). GC repression by a direct physical association between GR and NF-B was supported by several research groups, but these conclusions relied on in vitro data (226, 227, 228). Only recently, Adcock et al. (229) succeeded in showing an interaction between endogenous p65 and GR, using IL-1ß- and dexamethasone (DEX)-costimulated A549 cells, which contain a considerable amount of immunoreactive GR. It remains to be investigated whether such a complex is also formed during GR-mediated repression in other cell lines, whether ligand binding can play a modulatory role, and whether other factors or modifications are also involved. To further understand how GR interferes with the activity of NF-B and AP-1, several groups focused on delineating the relevant domains by mutation analysis or domain swapping experiments. Essentially, exchanging the DBD between different NRs (viz. GR, ER, and TRß) has proven the importance of the GR DBD both in transactivation and transrepression (102, 211, 230). Deleting the ligand-binding domain (LBD) diminished transrepression, whereas replacing it with an unrelated ß-galactosidase moiety greatly restored the transrepressive action, arguing for an exclusively steric role of the LBD (231). However, depending on the cell type and/or the NF-B-dependent promoter tested, some conflicting results were found regarding the requirement of the GR DBD (232) or the C-terminal zinc finger in NF-B transrepression (211, 233). The presence of a different subset of cofactors or GR function-modulating chaperones, or distinct signaling mechanisms in the different cell lines may explain particular discrepancies (1, 234, 235). Alternatively, the promoter context or effector site may also determine whether a specific NR can interfere with NF-B activity (236, 237, 238). NF-B-dependent up-regulation of ICAM-1 in human tracheal smooth muscle cells was found to be largely refractory to DEX inhibition, whereas simultaneous NF-B stimulation of the COX-2 gene did respond to the inhibitory action of DEX (239). Similarly, GR-mediated NF-B repression was found to be highly dependent on the core promoter and/or TATA-box environment (240, 241). For some hepatic acute-phase reactant genes, e.g., angiotensinogen, it appears that NF-B and GR positively interact at the acute phase response element to activate transcription (242, 243, 244).

Complementary to mapping the GR domains involved in NF-B repression, domains of p65 important in repressing the GR activity have also been mapped (245). Extensive mutational analysis illustrated that both the N-terminal RHD and the C-terminal domain of p65 are required for repression of GR transactivation. In vitro, a physical interaction could be demonstrated between GR and the RHD of p65, but not with the C-terminal part of p65 (228, 245). p50 Has also been shown to interact in vitro with GR, supporting the notion that there is an interaction with the homologous RHD. However, because p50 lacks transactivation domains, it cannot, in contrast to p65, reciprocally repress the transcriptional activity of the GR (228). Remarkably, c-Rel, which does contain a transactivation function, is also incapable of inhibiting GR-mediated transactivation. These data suggest that the presence of a conserved RHD alone is not sufficient to mediate repression and that an additional input is given by the unique transactivation functions of p65 (227).

Although AP-1 transrepression displays a lot of similarities to NF-B repression, some important differences are to be noted. Recently, a GR mutated in the first zinc finger (S425G) of the GR DBD was found to lose its capacity to repress NF-B without affecting AP-1 transrepression (246), allowing discrimination between both types of repression. Along the same line, the GC antagonist ZK98299 is not able to repress NF-B activity, whereas it efficiently inhibits AP-1 (211, 247). Repression specificity toward NF-B, AP-1, or other GR targets may be codetermined by distinct signaling mechanisms toward the various transcription components (see Section II.E.4, 7, and 9). Similarly, as for NF-B, repression of AP-1 activity was also shown to be strictly dependent on promoter, receptor, and cell type (248, 249).

C. Up-regulation of IB-
The alteration or induced expression of a regulatory protein capable of inhibiting NF-B activity may lie at the basis of GC repression of NF-B-mediated gene expression. One such candidate is the cytoplasmic inhibitor of NF-B, viz. IB-. GC-dependent repression of NF-B-driven genes has been proposed to be mediated by increased synthesis of IB-, which would then sequester NF-B in an inactive cytoplasmic form (Fig. 1G) (250, 251). However, the involvement of this mechanism cannot be generalized and seems to be strongly cell type and target gene dependent (Tables 3 and 4). Interestingly, other antiinflammatory signaling pathways (i.e., TGF-ß, IL-10, etc.) that inhibit NF-B activity through up-regulation of the IB- protein have also been described (8, 252, 253, 254, 255).

View this table: [in this window] [in a new window]   TABLE 3. Presence of up-regulation of IB- in GC-mediated repression

View this table: [in this window] [in a new window]   TABLE 4. Absence of up-regulation of IB- in GC-mediated repression

1. Transcriptional regulation of the IB- promoter by GCs.

The mechanism by which DEX stimulates the IB- promoter is still unresolved. The pIB--Luc (-623 to +11) promoter construct, transiently transfected in HeLa cells and induced with tetradecanoylphorbol acetate, showed a two-fold increase in luciferase activities when DEX was included (256); this is in agreement with data previously obtained in HeLa cells (250). Mutational analysis demonstrated that homodimerization of the GR is a prerequisite for induction of the IB- gene, which would argue for a classical GRE in the promoter (256). The same response element is also recognized by PR, in accordance with the fact that progesterone can also induce IB- synthesis (256). However, AR and ER are not able to enhance IB- synthesis in LNCaP prostate cancer cells and MCF-7 cells, respectively (256), or in AR-transfected COS-1 cells (257). On the other hand, an androgen-mediated increase in IB- synthesis was reported with endogenously present AR in LNCaP cells (258). The reason for these discrepancies remains unresolved. The suggestion of direct binding of GR to the IB- promoter DNA is complicated by the fact that no classical GRE can be detected up to 600 bp upstream of the start site of transcription. However, a related motif at positions -93 to -73 with a conserved one half of the normally palindromic hexanucleotide motif AGTTCT might suffice to carry out this induction (256). It would therefore be interesting to test the functionality of this putative GRE in HeLa cells by mutational analysis. Detailed DNase I footprinting recently confirmed a GR half-site at position -91/-81, although the results were obtained in breast cancer cells overexpressing GR (259).

The IB- promoter also contains three elements responsive to NF-B, which ensures a negative feedback loop for activation of NF-B. It is intriguing why this promoter does not show repression by DEX as observed with other NF-B-dependent promoters. In fact, a stably integrated pIB--Luc (-623 to +11) construct in L929 sA cells showed no enhancing effect of DEX alone or DEX + TNF on promoter activity, but was clearly repressed (202). Likewise, the porcine IB- promoter construct -600 to +20 coupled to luciferase and transiently transfected in BAEC cells showed induction with LPS or TNF, but was not induced by DEX (260). The basis for the apparent cell-specific opposing responses may be a cell-specific subset of cofactors (261, 262) that may allow the GR to cooperate, perhaps even in a DNA-binding independent way, with other LPS- or TNF-activated transcription factors in the IB--promoter. This type of regulation is not without precedent, because a cooperative effect between the GR and NF-IL6 has previously been demonstrated for activation of the ₁-acid glycoprotein gene (263, 264). Also, induction of the c-IAP2 promoter (containing two NF-B response elements and one GRE) by DEX and TNF results in a more than additive increase of the promoter activity. A c-IAP2 promoter variant in which the GRE site had been mutated resulted not only in loss of GC-mediated induction, but also, surprisingly enough, in loss of GC repression of the NF-B activity (238). In addition, synergistic stimulation of the IB- promoter can also be observed under conditions of activated NF-B and peroxisome proliferator-activated receptor PPAR- or the retinoid-related orphan receptor (ROR)- (265, 266). Interestingly, PPAR- ligand-dependent recruitment of vitamin D receptor-interacting protein (DRIP)/thyroid receptor-activated protein (TRAP) complex together with Sp1-flanking NF-B lies at the basis of the observed transcriptional synergy (265, 267). Whether this mechanism can be generalized for the GR and/or other cell types needs to be investigated further (268). The diversity of NR interactions with cofactor complexes may further be codetermined by chaperone proteins (107, 111, 112, 113, 154, 269, 270).

2. IB- expression vs. NF-B/DNA binding.
Conflicting results have been published on the relationship between IB expression levels and NF-B/DNA binding. A few groups found an elevated IB- protein level after a combined treatment with DEX and an inflammatory stimulus, concomitantly with a redistribution of p65 from the nucleus to the cytoplasm and a reduction in NF-B/DNA-binding, deemed responsible for gene repression (Fig. 1H and Table 3). In complete contrast, we and others observed DEX-mediated repression in the complete absence of IB- induction, without release of the TNF-induced NF-B complex from its response element in various cell types (Table 4). Similar observations were recorded for another NR, viz. the PR, which also antagonizes NF-B activity. This indicates that NRs can repress DNA-bound NF-B via tethering, without actually affecting DNA binding itself. In vivo footprinting experiments of the NF-B site in the ICAM promoter further proved that GC repression occurs by changing the conformation of the protein complex binding to the NF-B-binding site, without apparent perturbation of NF-B binding (87, 271). Sustained NF-B/DNA binding and resynthesis of IB may coexist if resynthesized IB is simultaneously degraded (272). Finally, repressive effects of DEX have also been described to appear with increased IB levels (but without a parallel decrease in NF-B/DNA binding) or with unaffected IB levels (with decreased NF-B expression levels) (Tables 3 and 4).

Intriguingly, in the neuronal cortex of DEX-treated rats, the levels of IB- are lower than in untreated animals, whereas the levels of IB- are enhanced in peripheral cells from the same animal. It would be interesting to investigate the underlying basis and the reason for the variations observed between related cell types in the same animal (273, 274). Apparently, there is no exclusive relationship between NF-B relocalization from nucleus to cytoplasm, reduced NF-B/DNA binding, and elevation in expression levels of IB- during GC repression.

3. Discriminating conditions determining a possible up-regulation of IB- by GCs.
Tables 3 and 4 show that IB- up-regulation is predominantly and consistently observed in lymphocytes and monocytes, whereas no such mechanism can be retrieved for endothelial or fibroblast cells in vitro. How can GCs achieve an up-regulation of IB- in some cell types and not in others? Different cell types may use alternative pathways to mediate GC effects. For example, in cells of lymphoid origin unique redox-sensitive NF-B signaling pathways requiring lipoxygenases or glutathione have been described (275, 276). In this respect, GC effects on oxidative stress and on lipoxygenase and glutathione levels have already been demonstrated for cells of lymphoid origin, arguing for the fact that unique redox-sensitive modes could have developed during evolution that may affect IB stability (277, 278, 279, 280, 281). Along the same line, JNK has been reported to mediate degradation of IB in a redox-dependent manner (282); because GCs were found to block JNK activity, IB- degradation may similarly be delayed (283, 284, 285, 286). Further evidence for this concept is provided by the fact that various links between IKK and JNK signaling have now been established (287, 288).

Besides cell-dependent variations in particular redox pathways, sensitivity to GC-induced apoptosis is also a cellular response known to be highly cell type- and stimulus-dependent (125, 289, 290). Cellular injury induces a differential adaptive response depending on the nature of the insult, whether physical (e.g., heat, radiation), chemical [e.g., reactive oxygen species (ROS), GCs], infectious (e.g., bacteria), or inflammatory (e.g., LPS, TNF). Recent data indicate that the cross-talk between various responses is not predictable and that permutations in triggering can have opposite effects on the outcome after injury (291, 292). For example, although it is well known that a prior heat shock can protect cells against inflammatory stress both in vitro and in vivo, it has also been shown that induction of a heat stress in cells primed by inflammation can precipitate cell death by apoptosis. This ability of heat shock to induce cytoprotection and cytotoxicity is therefore also known as the heat shock paradox. Experimental data currently link the heat shock paradox to induction of the NF-B inhibitor IB (293). Indeed, hsp proteins have currently been found to connect death receptor signaling, steroid activities, and inflammatory responses (112, 157, 160, 294, 295, 296, 297, 298, 299, 300, 301); besides its chaperone function in GR activity, hsp90 was recently found to be a functional component of the IKK complex, required for TNF signaling (302, 303, 304). Whether GC treatment relocates hsp90 association from GR to IKK complexes remains to be demonstrated, but this might explain why GCs modulate IB levels in particular cell types (112).

Besides hsp, ras chaperone proteins, proteasomes, and caspases have also been described as targets for GCs, which may in turn affect IB- turnover rates (169, 305, 306, 307). As such, GR/Raf1-Ras signaling toward a subclass of ras chaperone proteins was found to affect IB half-life (306, 307, 308). Proteasome inhibitors were found to sensitize leukemia cells for GC therapy (309). Furthermore, IB has been described as a caspase target both in vitro and in vivo (63, 70), whereas various caspases are required to mediate GC effects during apoptosis (310, 311, 312). Finally, differences in cell-culturing conditions and cell proliferation rate have been found to induce variations in GC-induced IB gene expression, depending on gene clusters involved in energy metabolism (313, 314).

From another point of view, cell culture experiments in vitro may not exactly reflect GC effects in vivo. In vascular endothelial tissue from patients suffering from Crohn’s disease, elevated levels of IB- were found after GC treatment, whereas in mononuclear cell infiltrates no such GC-induced up-regulation could be demonstrated (315). It was therefore concluded that up-regulation of IB- in these endothelial cells might correlate with the beneficial effects of GC treatment in Crohn’s disease. One should consider that, in chronic inflammatory disease models in vivo, the continuous induction of proinflammatory responses as well as the treatment last much longer (days to months) than investigations performed in in vitro cell lines (minutes to hours). In addition, in in vivo situations, many more parameters have to be taken into account. This includes signal transduction cascades elicited by different cell-cell contacts, systemic signals, GR metabolism, and neuroendocrine effects (178, 203, 316, 317).

4. Are GC-mediated transrepression and IB- up-regulation uncoupled phenomena?
The aforementioned observations raise the assumption that up-regulation of the IB- protein is not the main mechanism by which GCs can suppress immune genes. This view is further corroborated by various genetic approaches. First, the DNA-binding capacities of the GR itself do not determine transrepression, arguing against the induction by DEX of IB- as an element in transrepression (227). Furthermore, a dimerization-defective mutant rat GR (D4X, with the exchanges N454D, A458T, R460D, and D462C) (247) that does not bind DNA and does not transactivate GC-responsive genes or enhance IB- synthesis is still able to repress NF-B activity. These results have now been confirmed by experiments using mice with a dimerization-defective GR^dim/dim mutant (A458T), which demonstrates that GR/DNA binding and IB gene activation are dispensable for the antiinflammatory activity of the GR (197, 318, 319, 320). Reciprocally, the GC analogs ZK57740 and ZK077945, selected for their lack of antiinflammatory activities in vivo, do not repress NF-B-regulated genes but can still enhance IB- synthesis (256). Similar results were obtained with a GR mutant (S425G) lacking NF-B-repressing activity, but leaving enhanced IB synthesis intact (246). Second, repressive effects by the GR remain apparent in the presence of the protein synthesis inhibitor cycloheximide (321, 322, 323). Third, experiments with the GC antagonist RU486 or dissociated compounds RU24782 and RU24858 lacking GR transactivation activities demonstrated that GR-mediated transcription is not required for the inhibition of p65 transactivation (202, 228, 245). Moreover, the activity of constitutively nuclear Gal4-p65 chimeric proteins can efficiently be repressed by GCs, demonstrating that repression can occur in a promoter-independent way (322). Along the same line, a study comparing the activity of various clinically important GCs showed that it is possible to prevent TNF-induced degradation of IB- to various extents without affecting the NF-B/DNA-binding activity (324). Finally, comparable GC repression of NF-B has been observed in wild-type and IB-^-/- mouse embryonic fibroblasts (325, 326). These findings demonstrate that up-regulation of IB- and the phenomenon of GC repression are in many cases two independent processes.

If GC repression of NF-B activity and GC-mediated up-regulation of the IB- protein are uncoupled phenomena, the question remains what the biological significance is for the latter event. That two independent mechanisms of NF-B repression by GR may exist within the same cell suggests that maintaining negative control on NF-B-signaling pathways is of real physiological importance. IB- up-regulation represents a roundabout route to achieve effective repression, whereas a direct interference between preexisting, activated GR and NF-B proteins is a direct and quicker way to immediately repress proinflammatory excesses. The need for induction of IB- could, for instance, provide a molecular explanation for the limited efficacy of GCs in the therapy of septic shock (327). DEX-induced up-regulation of IB- has mainly been described for monocytes and T-lymphoid cells, which are sensitive to GC-induced apoptosis. In this respect, GCs are frequently used as therapeutic agents in the treatment of B or T cell lymphomas (328, 329, 330). Alternatively, in T cells, stimulation of IB- in response to GCs could have evolved to counter the antiapoptotic effects of constitutive NF-B levels by reducing its DNA binding (331). The first genetic evidence for NF-B in antiapoptotic events was found in p65-deficient embryos dying from massive liver apoptosis (33, 332, 333, 334). Analysis of mice carrying a dimerization-defective GR highlighted the importance of gene-inducing effects for subsequent apoptosis (197, 320). Interestingly, IB- induction was found in GC-induced apoptosis-sensitive cells, but not in resistant human leukemic T cells (335). Along the same line, variations in GC sensitivity and IB induction may also be caused by variations in GR/GRß ratio (336, 337). Overall, these data imply that particular cell types (such as T lymphocytes) need, in order to survive, threshold levels of NF-B transcriptional activity to maintain cell cycle progression (338, 339, 340, 341). This threshold may be subject to modulation by GCs via regulation of IB- expression during apoptosis (342, 343). This feedback mechanism may act as a back-up or final checkpoint to efficiently induce apoptosis in cells that sensed too much damage and to prevent an avalanche of systemic immune responses capable of inducing a life-threatening septic shock.

D. Cofactor competition model
Coactivator molecules are characterized by an intrinsic histone acetyltransferase (HAT) activity, believed to result in a more relaxed chromatin environment, which promotes gene activation (344). Hence, it may be assumed that competition between nuclear transcription factors for limited amounts of coactivator molecules leads to gene repression. The NR LBD has been shown to interact, in a ligand-dependent way, with coactivator proteins such as CBP, p300, and steroid receptor coactivator (SRC)-1 (345, 346). Because the same coactivators are also implicated in bridging p65, AP-1, or GR to the factors of the basal transcription machinery (347, 348, 349, 350, 351), transrepression was suggested to result from a competition between different transcription factors for a limited amount of cofactors (Fig. 1I). This model was first investigated for RAR- and GR-mediated repression of AP-1-dependent transactivation (347) and was supported by data from a number of other groups investigating negative cross-talk between various transcription factors and NRs (352, 353, 354, 355). Similarly, a competition between p65 or AP-1 and GR for limiting amounts of CBP or SRC-1 was proposed to account for transrepression of NF-B- and AP-1-dependent genes, respectively (356, 357, 358). However, a number of experiments and arguments counter the involvement of cofactor squelching in transrepression. First, an increase in coactivator concentrations (CBP, p300, SRC-1) in the cell generally leads to an increase in absolute gene expression levels of NF-B- or AP-1-driven promoters (which, in the presence of GR, was misinterpreted as relief of repression), but relative levels of GR-mediated transrepression remain unaffected. Notably, under conditions of GC repression, the physical association between p65 and CBP is not disrupted by repressing amounts of activated GR, both in vivo and in vitro (224, 240, 359). Second, if NR-mediated repression of both AP-1 and NF-B activities occurs through a general squelching for common cofactors, then RAR should also be able to mediate repression of NF-B. However, this NR only represses AP-1 activity, disfavoring a general competition model (360). Third, the existence of dissociating ligands (200, 202) as well as the availability of various receptor point-mutants of GR, which either separate transactivation and transrepression (197, 211, 320) or distinguish between NF-B and AP-1 repression (246), is not compatible with competition for a general cofactor (361, 362). Actually, GR may adopt a different conformation when working as a monomer in "trans" to inhibit NF-B activity or when it is bound to DNA as a homodimer to transactivate (319, 320, 363, 364, 365, 366, 367), requiring different cofactor configurations. In this respect, ligand-dependent allosteric effects of DNA-bound GR have recently been observed (368). Fourth, mutants of AP-1 that lack the N-terminal transactivation domain still repress NRs, whereas the interaction with CBP is lost (95, 96). Along the same line, the NF-B mutant Ser276C, defective in CBP recruitment (76), is as efficiently repressed as the wild-type molecule (240). In contrast, DNA-binding deficient mutants of p65, but with an intact predicted coactivator-recruiting transactivation domain, could no longer repress GC-mediated transactivation (245). These results suggest that competition for common cofactors is probably not a valid mechanism underlying mutual repression between GR and p65 or AP-1 (369). Finally, because various transcription factor families converge to the level of CBP/p300 for their transcriptional activities, the competition model struggles with a lack of specificity. If a cell were to inactivate the entire cellular pool of a given coactivator or activator in response to one signal, such a mechanism would preclude responsiveness by other activators or cooperativity at other genes in response to additional signals. As such, posttranslational modifications (e.g., phosphorylation, acetylation, methylation) (370, 371, 372, 373, 374, 375) or accessory chaperone proteins (e.g., SNIP-1, INHAT, DREAM, p35^rsj) (376, 377, 378, 379, 380) may selectively regulate cofactor access for specific transcription factors. Alternatively, CBP access may depend on dynamic nucleosome positioning around the target promoter of interest (381, 382, 383, 384, 385, 386).

Today, a number of observations are more consistent with the notion of territorial subdivision rather than a competition for factors (387, 388, 389, 390, 391). If transcription factor complexes are assembled within segregated nuclear compartments, then cofactor effects may be restricted to the designated compartment without affecting the same factors in other compartments associated with different genes (391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401). A specific nuclear matrix targeting signal has been identified within GR, including part of its DBD and transactivation domains (402, 403, 404). In addition, sumoylation, proposed to play a role in protein targeting, has now been observed for NF-B/IB (73, 74) as well as for GR (167, 170, 171). Of special interest is the cytoplasmic sequestration of nuclear corepressor (NCoR) and silencing mediator of retinoid and thyroid receptors (SMRT) corepressors upon complexation with IB/p65 RHD (405, 406). Nucleocytoplasmic shuttling is finally also affected by cofactor phosphorylation (407, 408).

Besides the spatial dimension of transcription, temporal aspects also argue against the cofactor competition model. Biological systems are highly dynamic, and transcription factors only transiently associate with their cognate DNA recognition sites and cofactor targets (368, 409, 410, 411, 412). In contrast to static transcription models supporting ordered recruitment of huge coregulator complexes (372, 413, 414, 415, 416, 417), more recent views propose very dynamic cofactor modules [(dis)assembly of distinct configurations depends on hsp chaperone molecules] that hit the promoter in a cyclic way during transcription (111, 112, 113, 418, 419). One study surprisingly revealed that ligand-dependent promoter remodeling, coactivator association, and target gene transcription induced by NRs are remarkably transient (minutes), despite continuous receptor association with the target DNA (hours) (420, 421, 422). Importantly, at a fixed DNA concentration, DEX-bound GR dissociates from DNA 10 times faster than does ligand-free GR or RU486-bound GR (368). Various experimental approaches (such as transient transfection, microinjection), which overload cells with transcription components (transcription factors, cofactors) neglect the dynamic stoichiometry of cofactor complexes and may not reflect appropriate regulation with respect to nuclear architecture (391, 393, 419, 422, 423, 424, 425). New RNAi approaches combining multiple somatic knockouts of transcription components in a single cell may soon shed new light on various aspects of NR and coregulator functions (235, 422).

E. New perspectives
1. Histone vs. (co)factor acetylation.
Because simple competition for common coactivators is probably not the main mechanism of GC repression, the question remains what the effective mechanism is. As an alternative to cofactor competition, a coactivator repulsion model, based on transcription factor domains that prevent enhanceosome-dependent recruitment of the CBP-PolII holoenzyme complex by repulsion, was suggested (415, 426). However, we and others found no disruption of p65-CBP interaction under repressive conditions with the GR (240, 427). Over the last 10 yr, a vast amount of novel proteins interacting with members of the NR superfamily were identified by two-hybrid screening, functional complementation studies, far-Western blotting, and expression cloning (101, 262). Most of these proteins appear to be ubiquitously expressed and to interact with multiple members of the NR superfamily, although specificities and different affinities have also been detected (261, 268, 428, 429, 430, 431, 432). It should be noted that a correlation between levels of histone acetylation and transcriptional activity of specific loci has been established (433). Similarly, targeted deacetylation of chromatin may contribute to transcriptional repression in mammals (434, 435). Some members of nuclear hormone receptors, such as TR, actively silence gene expression in the absence of hormone. Corepressors, which bind to the receptors silencing domain, are involved in this repression (436, 437). A histone deacetylase (HDAC)-containing corepressor complex consisting of NCoR, SMRT, mSin-3, and RPD-3/HDAC-1 was identified to be associated with unliganded RAR/RXR and TR (438, 439). Upon ligand binding, this silencing complex is displaced by a HAT-containing coactivator complex comprising CBP, p300/CBP-associated factor (p/CAF) and SRC-1 (440, 441). Thus NR-dependent transcription may be regulated by an acetylation/deacetylation flip-flop mechanism (442, 443) (Fig. 1J). Of particular interest is the possibility that multiple ligands for NRs influence the biological activity of the receptor by selectively affecting the recruitment of coregulator complexes (361, 362, 397, 444, 445, 446). Cocrystal structures have revealed that antagonist-bound and agonist-bound ER display a different position of helix 12 in the LBD (447, 448). Similarly, antagonist-bound PR was shown to interact in vitro with the corepressor NCoR (449). Furthermore, NCoR and SMRT associated only with antagonist-bound PR and ER, as assessed by a two-hybrid screen (450, 451, 452). A novel coregulatory protein, template-activating factor Iß associates with ER and regulates transcription of estrogen-responsive genes by modulating acetylation of histones and ER (453). In a molecular dynamics study, it has recently been shown that the GR DBD can exist in two conformational states, a transcriptionally active and a transcriptionally inactive state (454). The transactivating DNA-bound homodimeric GR may, as opposed to the repressing non-DNA-bound monomer, adopt a different conformation, favoring interactions with NR coactivator or corepressor complexes (363, 365, 368). In this respect, the crystal structure of the human GR LBD, bound to DEX and a coactivator motif, derived from the transcriptional intermediary factor 2 (TIF-2), adopts a surprising dimer configuration involving formation of an intermolecular ß sheet; an additional charge clamp determines the binding selectivity of cofactors, whereas a distinct ligand-binding pocket explains its selectivity for endogenous steroid hormones (198, 364, 455). The synergism between GR and c-Jun homodimers is not easily explained; it would require a GRE-bound GR conformation in a composite element context. The allosteric model does, however, not suffice to explain why the nontransactivating form of GR actively hinders the activity of the Jun/Fos heterodimer (456), unless one assumes that a GR-bound corepressor molecule can also negatively influence the neighboring Jun/Fos heterodimer. An important challenge for future experiments will be to provide the currently lacking experimental connection between in vitro data (overexpression) and in vivo behavior of the receptor [chromatin immunoprecipitations, real-time imaging by means of green fluorescent protein (GFP), fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), bimolecular fluorescence complementation (BiFC), etc.] with respect to its cofactor partners (274, 392, 457, 458). The physiological relevance of predominantly in vitro observations can ultimately be answered only in knockout mice of individual coactivators, like that of SRC-1 (459), or in combined somatic knockouts (e.g., NCoR, SMRT, SRC-1, CBP) by means of RNAi (235, 460).

There is no doubt that GR will recruit specific coactivators to enable transactivation. The key question to be addressed is whether a distinct GR cofactor configuration is involved in repression of NF-B-mediated gene expression (361, 362, 443, 461). Recently, GR has been found to be associated with HDAC-2 in vivo. In addition, GR antagonist was able to abrogate this interaction (462). Blocking HDAC-2 activity by cigarette smoke in alveolar macrophages was further found to block GR transrepression and increase cytokine expression (463). Interestingly, HAT and HDAC activities coexist within the same complex in the presence of p65 and GR, and they can each act independently without competing with each other, as revealed by in vivo chromatin immunoprecipitations (77, 427). A different histone acetylation pattern was observed in the presence of p65 alone, as compared with p65 and GR. In addition, GR was able to block specific histone acetylation and CBP phosphorylation under particular conditions, which may be tightly linked to gene repression (427, 464, 465, 466). In this configuration, the HDAC inhibitor trichostatin A (TSA) again relieves GR-mediated repression. However, similarly as for CBP overexpression experiments, reporter gene activities in response to the GR ligand DEX+TNF+TSA should be compared with the response to TNF+TSA, demonstrating that relative repression is conserved under conditions of inhibited deacetylases (357, 427, 462, 463). In addition, promoter responsivity to TSA does not necessarily reflect sensitivity to GCs because IL-8 and HIV promoter activity can be similarly increased with TSA, whereas only the IL-8 promoter shows a strong repression in the presence of DEX. This proves that the dynamic balance of acetylation/deacetylation can be uncoupled from GR-mediated repression (224). It still remains to be established how liganded GR recruits HDAC-2 to the p65-CBP HAT complex. Besides HDAC-2, association of NF-B with HDAC-1 and HDAC-3 has also been observed recently (77, 467, 468). Because histones (465, 469, 470), NRs (172, 173), NF-B (468), as well as cofactors (370, 371, 420, 471) can be (de)acetylated, it will be interesting to understand cross-talk of the various modifications under conditions of gene activation and GC repression (372, 442, 472, 473, 474).

2. Methylation of histones, (co)factors and DNA.
Besides acetylation, other posttranslational modifications suc